—Cardiac arrhythmia is a common and often lethal manifestation of many forms of heart disease. Gap junction remodeling has been postulated to contribute to the increased propensity for arrhythmogenesis in diseased myocardium, although a causative role in vivo remains speculative. By generating mice with cardiac-restricted knockout of connexin43 (Cx43), we have circumvented the perinatal lethal developmental defect associated with germline inactivation of this gap junction channel gene and uncovered an essential role for Cx43 in the maintenance of electrical stability. Mice with cardiac-specific loss of Cx43 have normal heart structure and contractile function, and yet they uniformly (28 of 28 conditional Cx43 knockout mice observed) develop sudden cardiac death from spontaneous ventricular arrhythmias by 2 months of age. Optical mapping of the epicardial electrical activation pattern in Cx43 conditional knockout mice revealed that ventricular conduction velocity was significantly slowed by up to 55% in the transverse direction and 42% in the longitudinal direction, resulting in an increase in anisotropic ratio compared with control littermates (2.1±0.13 versus 1.66±0.06; P <0.01). This novel genetic murine model of primary sudden cardiac death defines gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate.

Integrins regulate cellular behaviors through signaling pathways, including Rho GTPases and kinases. CD98 heterodimers, comprised of a heavy chain (CD98hc, SLC3A2) and one of several light chains, interact with integrins through CD98hc. CD98hc overexpression leads to anchorage-independent cell growth and tumorigenesis in 3T3 fibroblasts and activates certain integrin-regulated signaling pathways. To establish the biological function of CD98hc, we disrupted the gene and analyzed CD98hc-null cells. Here we report that CD98hc contributes to integrin-dependent cell spreading, cell migration, and protection from apoptosis. Furthermore, CD98hc is required for efficient adhesion-induced activation of Akt and Rac GTPase, major contributors to the integrin-dependent signals involved in cell survival and cell migration. CD98 promotes amino acid transport through its light chains; however, a CD98hc mutant that interacts with β1 integrins, but not CD98 light chains, restored integrin-dependent signaling and protection from apoptosis. β1 integrins are involved in the pathogenesis of certain cancers. CD98hc deletion markedly impaired the ability of embryonic stem cells to form teratocarcinomas in mice; teratocarcinoma formation was reconstituted by reexpression of CD98hc or of the mutant that interacts exclusively with integrins. Thus, CD98hc is an integrin-associated protein that mediates integrin-dependent signals, which promote tumorigenesis.

We have investigated the block to expression of Moloney murine leukemia virus in murine embryonal carcinoma (EC) cells. Infected EC cells were found to contain up to 100 integrated proviral genomes. However, expression of virus as measured by XC plaque and virus-specific RNA synthesis did not occur at significant levels, in contrast to productively infected differentiated cells. Analysis of the DNA in the infected EC cells revealed that the proviral genomes were highly methylated, as shown by their resistance to cleavage by Sma I. Integrated proviral genomes in infected differentiated cells were readily cut by Sma I and thus were not methylated at these sites. Transfection of DNA from infected EC cells to cells permissive for virus expression failed to induce virus expression. The proviral genomes, however, were potentially infectious because they induced XC plaques when the recipient cells for transfection were treated with 5-azacytidine. This drug is believed to interfere with DNA methylation. We conclude that expression of proviral genomes introduced into EC cells is suppressed and that this inactivation can be correlated with the de novo methylation of the viral DNA. De novo methylation activity thus may be a characteristic of early embryonic cells.